The newly derived cell line Rauscher murine erythroleukemia undergoes erythroid differentiation in response to erythropoietin as well as synthetic chemical inducers. Using clonal lines of these cells we have shown that the responses to these inducers are selective and can be segregated. These cells develop into enucleate, mature-appearing erythrocytes when exposed to erythropoietin in suspension culture. Thus, they represent an ideal model system for the investigation of the cellular physiology and biochemistry of erythropoiesis. We propose to clone the primary line on a large scale and to derive sublines which exhibit selective responses to erythropoietin, dimethylsulfoxide, butyric acid and hemin. The morphology, growth characteristics, karyotype and phenotypic stability of each line will be established. We will analyze the degree of complete hemoglobin synthesis and specific globin chain synthesis of selected clones as a function of inducer in order to explore potential approaches to the modulation of globin gene expression and hemoglobin switching. The sequence of expression of four erythroid markers globin mRNA, transferrin receptors, membrane spectrin and uroporphyrinogen I synthetase will be established in order in ask 1) is the sequence variable among different clones, 2) does it vary with inducer, 3) can some steps in the program be skipped? Furthermore we will begin to elucidate the mechanism of action of erythropoietin through examination of its effects on cell membrane potentials and cyclic nucleotides. The role of the cell cycle in erythropoiesis will be studied with synchronized cultures. This work will serve as a foundation for the elucidation of the molecular basis of erythropoietin's action and is of potentially great importance in understanding and ultimately controlling red cell production in both inherited and acquired anemias.